The present invention is in the field of microbiology and more particularly in the field of halophilic, alkaliphilic microorganisms.
Alkaliphilic microorganisms are defined as organisms which exhibit optimum growth in an alkaline pH environment, particularly in excess of pH 8, and generally in the range between pH 9 and 10. Alkaliphiles may also be found living in environments having a pH as high as 12. Obligate alkaliphiles are incapable of growth at neutral pH.
Alkaliphiles may be found in such everyday environments as garden soil, where transient alkaline conditions may arise due to biological activity such as ammonification, sulphate reduction or photosynthesis. A much richer source of a greater variety of alkaliphilic organisms may be found in naturally occurring, stable alkaline environments such as soda lakes.
Halophilic bacteria are defined as microorganisms that grow optimally in the presence of salt (sodium chloride). Since microorganisms are often capable of growth over a wide range of salt concentrations, the term halophile is usually reserved for microorganisms having a minimum requirement in excess of the concentration found in sea water (ca. 0.5 M or 3%).
Extremely halophilic bacteria are defined as bacteria that grow optimally at above 20% NaCl (3-4 molar). Extreme halophiles inhabit hypersaline environments. The most intensely studied extremely halophilic bacteria belong to the order Halobacteriales. With the exception of the genera Natronobacterium and Natronococcus, all known Halobacteria are obligate halophiles which require at least 12-15% salt for growth and a pH around neutrality. These bacteria belong to the Kingdom Euryarchaeota of the Domain Archaea (the archaeobacteria) (Woese, C. R., et al, Proc. Natl. Acad. Sci. U.S.A., 87, (1990), 4576-4579).
The term xe2x80x9chaloalkaliphilexe2x80x9d was first used by Soliman and Truper to describe bacteria that are both halophilic and alkaliphilic. (Soliman, G. S. H. and Truper, H. G., (1982), Zbl. Bakt. Hyg., I. Abt. Orig. C3, pp. 318-329). Until now the only known examples of such bacteria belong to the Kingdom Euryarchaeota (Tindall, B. J. and Truper, H. G., (1986), System. Appl. Microbiol., 7, 202-212).
The most extreme hypersaline environments are microbiologically the least diverse but nevertheless contain a distinct, rich and complex flora of extreme halophilic bacteria. It has been suggested that these environments are dominated by the Euryarchaeota with few eubacteria present (Rodriguez-Valera, F., in Halophilic Bacteria, vol. 1, (Rodriguez-Valera, F., ed.) CRC Press, Inc., Boca Raton, Fla., (1988), pp. 3-30).
Soda lakes, an example of naturally-occurring alkaline environments which may also be hypersaline, are found in various locations around the world. They are caused by a combination of geological, geographical and climatic conditions. They are characterized by the presence or large amounts of sodium carbonate (or complexes of this salt) formed by evaporation concentration, as well as the corresponding lack of Ca2+ and Mg2+ which would remove carbonate ions as insoluble salts.
In situations where the concentrations of Ca2+ and Mg2+ exceed that of carbonate, or where they are equimolar, a salt lake is generated with pH 6-8, and whose ion composition is dependent on the local geology. The Dead Sea in Israel is a typical example of a slightly acidic (pH 6-7) saline lake enriched with divalent cations, particularly Mg2+. On the other hand, the Great Salt Lake in Utah, U.S.A. is an example of a Mg2+-depleted brine and is slightly alkaline (pH 7-8).
The commercial production of common salt from sea water in solar evaporation ponds (salterns) generates man-made hypersaline environments. Salterns provide excellent model systems over a range of salinities (from sea water to super-saturation), and their chemistry and microbiology have been intensely studied (Javor, B., in Hypersaline Environments, Springer-Verlag, Berlin/Heidelberg, 1989).
The African Rift Valley is probably unusual in having lakes with significant, largely permanent bodies of brine. The Kenyan-Tanzanian section of the Rift Valley contains a number of alkaline soda lakes with a range of total salinities from around 5% (w/v) in the more dilute lakes (e.g. Elmenteita, Bogoria, Nakuru, etc.), to saturation (30% or greater) in parts of lakes Magadi, Little Magadi (Nasikie Engida) and Natron. These lakes are devoid of significant amounts of Ca2+ and Mg2+ (in most cases below the level of detection) and have pH values in the range from 9 to above 11.5 in the most concentrated lakes.
Despite this apparently harsh environment, soda lakes are nevertheless home to a large population of prokaryotes, a few types of which may dominate as permanent or seasonal blooms. The organisms range from alkaliphilic cyanobacteria to haloalkaliphilic archaeobacteria. At the higher salinities (characterized by higher conductivities) haloalkaliphilic archaeobacteria predominate. Moreover, it is not unusual to find common types of alkaliphilic organisms inhabiting soda lakes in various widely dispersed locations throughout the world such as in the East African Rift Valley, in the western U.S., Tibet, China and Hungary. For example, natronobacteria have been isolated and identified from soda lakes and soils located in China (Wang, D. and Tang, Q., xe2x80x9cNatronobacterium from Soda Lakes of Chinaxe2x80x9d in Recent Advances in Microbial Ecology (Proceedings of the 5th International Symposium on Microbial Ecology, eds. T. Hattori et al., Japan Scientific Societies Press, Tokyo, (1989), pp. 68-72), the Soviet Union (Zvyagintseva, I. S. and Tarasor, A. L. (1988) Microbiologiya, 57, 664-669) and in the western U.S. (Morth, S. and Tindall, B. J. (1985) System. Appl. Microbiol., 6, 247-250). Natronobacteria have also been found in soda lakes located in Tibet (W. D. Grant, unpublished observations) and India (Upasani, V. and Desai, S. (1990) Arch. Microbiol., 154, pp. 589-593).
A more detailed study of soda lakes and alkaliphilic organisms in general is provided in Grant, W. D., Mwatha, W. E. and Jones, B. E. (1990) FEMS Microbiology Reviews, 75, 255-270, the text of which is hereby incorporated by reference. Lists of alkaline soda lakes may be found in the publications of Grant, W. D. and Tindall, B. J. in Microbes in Extreme Environments, (eds. R. A. Herbert and G. A. Codd); Academic Press, London, (1986), pp. 22-54); and Tindall, B. J. in Halophilic Bacteria, Volume 1, (ed. F. Rodriguez-Valera); CRC Press Inc., Boca Raton, Fla., (1988), pp. 31-70, both texts are also hereby incorporated by reference. A detailed study of hypersaline environments is provided in Javor, B., in Hypersaline Environments, supra).
Alkaliphiles isolated from non-saline environments are also discussed by Horikoshi, K. and Akiba, T. in Alkalophilic Microorganisms (Springer-Verlag, Berlin/Heidelberg/N.Y., 1982). However, alkaliphilic organisms from saline environments such as soda lakes are not discussed therein. Strictly anaerobic bacteria from alkaline, hypersaline environments have been recently described by Shiba, H., in Superbugs (eds. K. Horikoshi and W. D. Grant); Japan Scientific Societies Press, Tokyo, and Springer-Verlag, Berlin, Heidelberg, N.Y., (1991), pp. 191-211; and by Nakatsugawa, N., ibid, pp. 212-220.
Alkaliphiles have already made an impact in the application of biotechnology for the manufacture of consumer products. Alkaliphilic enzymes produced by alkaliphilic microorganisms have already found use in industrial processes and have considerable economic potential. For example, these enzymes are currently used in detergent compositions and in leather tanning, and are foreseen to find applications in the food, waste treatment and textile industries. Additionally, alkaliphiles and their enzymes are potentially useful for biotransformations, especially in the synthesis of pure enantiomers. Also, many of the microorganisms described herein are brightly pigmented and are potentially useful for the production of natural colorants.
The present invention provides pure cultures of novel haloalkaliphilic bacteria. These bacteria have been isolated from samples of soil, water, sediment, trona (NaHCO3.Na2CO3.2H2O) and a number of other sources, all of which were obtained from in and around alkaline, hypersaline lakes. These haloalkaliphiles have been analyzed according to the principles of numerical taxonomy with respect to each other and also to other known haloalkaliphilic bacteria in order to confirm their novelty. In addition, these bacterial taxa are further circumscribed by chemotaxonomic analysis.
The present invention also provides data as to the composition of the environments from which the samples containing the microorganisms were obtained, as well as the media required for their efficient isolation and culturing such that one of ordinary skill may easily locate such an environment and be able to, isolate the organisms of the present invention by following the procedures described herein.
It is also an object of the present invention to provide microorganisms which produce useful alkali- and salt-tolerant enzymes, as well as methods for obtaining substantially pure preparations of these enzymes. These enzymes are capable of performing their functions in high pH, saline environments which makes them uniquely suited for applications requiring such extreme conditions. For example, enzymes having alkali- and salt-tolerance may be employed in detergent compositions, in leather tanning and in the food, waste treatment and textile industries, as well as for biotransformations such as the production of pure enantiomers.